The articles set forth in the Background of the Invention are incorporated herein by reference.
Capillary zone electrophoresis ("CZE") is a technique which permits rapid and efficient separations of charged substances. In general, CZE involves introduction of a sample into a capillary tube, i.e. a tube having an internal diameter from about 5 to about 2000 microns, and the application of an electric field to the tube. The electric potential of the field both pulls the sample through the tube and separates it into its constituent parts. Each constituent of the sample has its own individual electrophoretic mobility; those having greater mobility travel through the capillary tube faster than those with slower mobility. As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones.
CZE can be generally separated into two categories based upon the contents of the capillary columns. In "gel" CZE, the capillary tube is filled with a suitable gel, e.g., polyacrylamide gel. Separation of the constituents in the sample is predicated in part by the size and charge of the constituents travelling through the gel matrix. In "open" CZE, the capillary tube is filled with an electrically conductive buffer solution. Upon ionization of the capillary, the negatively charged capillary wall will attract a layer of positive ions from the buffer. As these ions flow towards the cathode, under the influence of the electrical potential, the bulk solution (the buffer solution and the sample being analyzed), must also flow in this direction to maintain electroneutrality. This electroendosmatic flow provides a fixed velocity component which drives both neutral species and ionic species, regardless of charge, towards the cathode. The buffer in open CZE is as stable against conduction and diffusion as the gels utilized in gel CZE. Accordingly, separations can be obtained in open CZE quite similar to those obtained in gel-based CZE.
Fused silica is principally utilized as the material for the capillary tube because it can withstand the relatively high voltage used in CZE, and because the inner walls of a fused silica capillary ionize to create the negative charge which causes the desired electrosomatic flow. The ionization of the inner walls of the capillary tube, however, creates problems with respect to separation of proteinaceous materials. Proteins are hetero-polyelectrolytes (i.e. an approximate equivalent number of positively and negatively charged moieties within the molecule while the molecule itself has a neutral-charge). Thus, when ionized, a protein species can have a net positive charge distribution such that the protein species will adsorb quite strongly onto the ionized inner wall. This adsorption leads to artificial zone broadening in CZE, resulting in inconclusive, erroneous or incomprehensible results.
The pH of the electrolyte buffer can dramatically effect the efficiencies and resolutions of separation by CZE. Even a small shift in pH can have a large impact on the separation. With untreated fused silica capillary columns, however, this fact is a double edged sword because pH's other than near-neutral lead to the formation of negatively charged silanol groups on the inner wall of the capillary. Thus, heretofore, untreated fused silica capillary columns could not be used with a wide range of pH values.
One proposed attempt at solving this problem was to treat, or "coat", the inner wall of the capillary tube so that electrosomatic flow would be reduced when voltage was applied. That would, in turn, reduce adsorption of proteins onto the tube. Glycol modified fused silica capillaries have been used for serum protein analysis, but only with limited success. See Jorgenson, J. W. & Lukacs, K. D. "Capillary Zone Electrophoresis." Science 222:266-272 (1983). U.S. Pat. No. 4,680,201 describes coated capillary tubes comprising a bifunctional compound having a first functional group covalently attached to the wall and a second functional group capable of being polymerized. See also, Hjerten, S. "High-Performance Electrophoresis Elimination of Electrophoresis and Solute Adsorption." J. Chrom. 547:191-198 (1985) and Cobb, K. A. et al "Electrophoretic Separations of Proteins in Capillaries with Hydrolytically Stable Surface Structures." Anal. Chem. 62:2478-2483 (1990). Other covalently attached coatings are described in U.S. Pat. No. 4,931,328 and PCT Published Application No. WO 89/12225. See also, Swedberg, S. A. "Characterization of Protein Behavior in High-Performance Capillary Electrophoresis Using a Novel Capillary System." Anal. Biochem. 185:57-56(1990) (hereinafter "Swedberg").
Concomitantly, coated fused silica capillaries have a relatively short shelf-life and their coatings have a tendency to "dissolve" in an unpredictable manner. The aura of unpredictability is unacceptable in any environment where multiple samples will be analyzed on a frequent basis. Aside from the practical limitations with coated capillary columns, the associated costs also make them impractical. A coated capillary column applicable to commercially available CZE analyzers costs approximately $90.00 (United States of America. Of this amount, approximately $1.00 (United States of America) is attributed to the cost of the fused silica capillary itself. Thus, the major cost of such commercially available columns is related to the coating itself. On average, coated columns will begin to deteriorate after about 50 to 100 runs. As such, they are expensive to use.
Another proposed solution to the problem of protein adsorption was to use a buffer having a pH greater than the isoelectric points (pI) of the protein components of the sample. As is well known, when the pH is equal to the pI, the positive and negative moieties of the molecule are balanced. Similarly, when the pH is greater than pI, the negative moieties predominate and when the pH is less than the pI, the positive moieties exceed the negative moieties. For example, the pI of albumin is 4.6; therefore, at pH 4.6, the negatively charged and positively charged moieties of albumin are equally distributed on the surface of the albumin molecule and its overall charge is neutral. However, as the pH is raised above the isoelectric point, the negatively charged moieties predominate and the net charge is negative. Thus, under the influence of a high pH buffer, all of the protein species of the sample will have a negative charge and will be repelled from the negatively charged wall. This will, in turn, avoid or at least greatly diminish, their surface adsorption. However, large pH-pI differences can cause structural changes in the protein, or even hydrolysis. Attempts to electrophorese complex mixtures such as, e.g. human serum protein, in untreated fused-silica capillary tubes using buffer solutions having pH ranges from 5-8 have resulted in irreproducible migration of all sample zones. See Lauer, H. H. and McManigill, D. "Capillary Zone Electrophoresis of Proteins in Untreated Fused Silica Tubing." Anal. Chem, 58:166-169 (1986).
It has been theorized that protein adsorption onto the untreated fused capillary wall is due to ion exchange interactions between cationic sites in the protein and silicate moieties in the wall. See Jorgenson, J. W. "Capillary Electrophoresis", Chpt. 13, New Direction in Electrophoretic Methods. ACS Symp. Ser. 335, 1987 (Jorgenson, J. W. & Phillips, M., Eds.). Accordingly, it has been suggested to use high salt buffer conditions to reduce protein adsorption. See Lauer, H. H. & McManigill, D., Trends Anal. Chem. 5:11 (1986). However, increasing the salt concentration of the buffer has the effect of increasing the conductivity of the capillary tube which can dramatically increase the heat inside the tube. Increasing temperature causes the migrating zones to become diffused, thus decreasing resolution of the zones. In order to avoid such heat build-up, the electric potential applied to the capillary tube must be greatly diminished. This, however, has the undesirable effect of increasing the time necessary for analysis of the sample.
Alkali metal salts have been added to buffers in an effort to minimize protein absorption on fused-silica capillary tubes. Green, N. S. and Jorgenson, J. W. "Minimizing Adsorption of Proteins on Fused Silica in Capillary Zone Electrophoresis by the Addition of Alkali Metal Salts to the Buffers." J. Chrom. 478:63-70(1989) (hereinafter "Green"). Addition of KS.sub.2 SO.sub.4 to a pH 9.0 buffer was reported to evidence little adsorption of two proteins which ordinarily demonstrate significant adsorption in a pH 9.0 buffer (lysozyme and trypsinogen). Similarly, zwitterionic salts have been added to such buffers. Busey, M. M. and Jorgenson, J. W. "Capillary Electrophoresis of Proteins in Buffers Containing High Concentrations of Zwitterionic Salts." J. Chrom. 480:301-310 (1989).
None of the preceding methodologies are sufficient for separating sample constituents over a wide range of pH values, i.e. about pH 3.0 to about pH 11.0. This is particularly highlighted in the untreated (i.e. non-coated) columns. As noted, each constituent of the sample to be separated has a unique isoelectric point. Thus, if the pH of the buffer is, e.g., 7.0, and the isoelectric points of two constituent samples are, e.g., 2.0 and 4.0, respectively, the resulting electropherogram may not evidence distinction between the two constituents. This is because the pH of the buffer may not allow for their proper separation, thus leading to co-migration of the two constituents which would appear as a single peak on an electropherogram.
Use of different buffer systems having different pH values has the undesirable effect of adding multiple variables to the analysis. I.e, an acidic pH (less than about 4.0) buffer may "interact" with the sample constituents in a manner differently than an alkaline pH buffer (greater than about 8.0). Ideally, a single buffer systems capable of having a range of pH values should be utilized such that any internal variability is negated.
Present coated capillary columns cannot withstand the rigors of buffers having the types of pH ranges noted above, i.e. from about pH 3.0 to about pH 11.0, due to the inherent unpredictability and instability thereof. Untreated columns avoid this problem, but have inherent problems with respect to sample constituent adsorption. What is needed, then, is a CZE buffer applicable over a range of pH values, which can be used in conjunction with open-tube CZE, and which substantially diminishes sample-constituent adsorption onto untreated capillary tubes.